Hereditary Multiple Exostoses (HME) is an autosomal dominant disorder that affects about 1 in 20,000 children. HME is characterized by cartilage-capped outgrowths that form adjacent to the growth plates, protrude into surrounding tissues and organs, and cause growth retardation, compression of nerves and early onset osteoarthritis. They become malignant in about 5% of the patients. Current therapies are palliative, and patients struggle with pain and limited mobility and undergo multiple surgeries through life. The genes responsible for HME cases are EXT1 and EXT2 that encode glycosyltransferases responsible for heparan sulfate (HS) synthesis. Patients are heterozygous for EXT1 or EXT2 loss-of-function mutations and their cells produce lower HS amounts. HS-rich proteoglycans regulate key physiologic processes by various mechanisms and most notably by restricting the topographical distribution and action of hedgehog, BMPs and other signaling factors within tissues, but it is not known whether defects in these mechanisms subtend HME. In ongoing studies, we found that HS deficiency in growth plate leads to re-distribution of Indian hedgehog (Ihh), its infiltration over the entire perichondrium and formation of exostosis-like cartilaginous masses within perichondrium itself. A similar ectopic action of Ihh was seen in mouse growth plates deficient in HS N- sulfation. We found also that interference with HS function greatly stimulates differentiation of mesenchymal cells into chondrocytes. Thus, our central hypothesis is that the HS deficiency in HME (i) causes re-distribution of hedgehog and other pro-chondrogenic factors from growth plate to perichondrium and (ii) enhances responsiveness of perichondrial cells to these and other local factors. As a result of this combination of mechanisms, growth plate and perichondrium would mis-communicate, and perichondrial cells would lose their normal character, become chondrogenic and give rise to exostoses. To test our hypotheses, we will analyze the mechanisms of exostosis formation by creating conditional Ext-deficient mice in growth plate and/or perichondrium and determining roles of pro-chondrogenic signaling pathways (Aim 1). We will determine the mechanisms for increased chondrogenic capacity of HS-deficient cells will test their responsiveness to signaling factors and assess structure and protein binding capabilities of their HS chains (Aim 2). We will then carry out proof-of-principle experiments to determine whether pharmacologic antagonists of pro-chondrogenic signaling pathways block exostosis formation (Aim 3). The project will provide fundamentally new insights into the cellular and molecular mechanisms of HME pathogenesis and will test possible rational therapies based on those insights. The project thus has significant importance for both basic biomedical research and translational medicine in HME and related growth plate-based skeletal dysplasias. The number of HME patients is small, but the community of their families is large. This project will thus provide a renewed sense of hope to patients and families alike that this neglected disease will actively be studied and a cure may one day be found. PUBLIC HEALTH RELEVANCE: Hereditary Multiple Exostoses (HME) is a serious disease that affects about 1 in 20,000 children and an adolescent, causes growth retardation, continuous pain, limited mobility and fatigue, and is associated with bone malignant tumors. There are no cures or effective treatments at the moment, and this project thus aims to identify the mechanisms of pathogenesis and test a specific therapeutic treatment to prevent formation of tumor-like bone (exostosis) typical of this disease.